John et al. Reproductive Biology and Endocrinology (2019) 17:53 https://doi.org/10.1186/s12958-019-0491-x

RESEARCH Open Access Comparative study of environmental pollutants and on sexual differentiation of anteroventral periventricular nucleus and spermatogenesis Naham John1, Humaira Rehman1, Suhail Razak1,2* , Mehwish David1, Waheed Ullah1, Tayyaba Afsar2, Ali Almajwal2, Iftikhar Alam2 and Sarwat Jahan1

Abstract Background: Bisphenol A is well known endocrine-disrupting chemical while Bisphenol S was considered a safe alternative. The present study aims to examine the comparative effects of xenobiotic bisphenol-A (BPA) and its substitute bisphenol-S (BPS) on spermatogenesis and development of sexually dimorphic nucleus population of dopaminergic neurons in the anteroventral periventricular nucleus (AVPV) of the in male pups. Methods: Sprague Dawley rat’s pups were administered subcutaneously at the neonatal stage from postnatal day PND1 to PND 27. Thirty animals were divided into six experimental groups (6 animals/group). The first group served as control and was provided with normal olive oil. The four groups were treated with 2 μg/kg and 200 μg/kg of BPA and BPS, respectively. The sixth group was given with 50 μg/kg of dissolved in olive oil as a standard to find the development of dopaminergic tyrosine hydroxylase neurons in AVPV regions. Histological analysis for testicular tissues and immunohistochemistry for brain tissues was performed. Results: The results revealed adverse histopathological changes in testis after administration of different doses of BPA and BPS. These degenerative changes were marked by highly significant (p < 0.001) decrease in tubular and luminal diameters of seminiferous tubule and epithelial height among bisphenols treated groups as compared to control. Furthermore, significantly increased (p < 0.001) TH-ir cell bodies in the AVPV region of the brain with 200 μg/kg dose of BPA and BPS was evident. Conclusion: It is concluded that exposure of BPA and BPS during a critical developmental period can structural impairments in testes and affects sexual differentiation of a dimorphic dopaminergic population of AVPV region of hypothalamus in the male brain. Keywords: , Bisphenol A, Bisphenol S, Immunohistochemistry, AVPV

* Correspondence: [email protected]; [email protected] 1Reproductive Physiology Lab, Department of Animal Sciences, Quaid- i- Azam University Islamabad, Islamabad 45320, Pakistan 2Department of Community Health Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Kingdom of Saudi Arabia

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. John et al. Reproductive Biology and Endocrinology (2019) 17:53 Page 2 of 10

Background alterations in an embryonic, nervous and endocrine sys- Bisphenol A (BPA) is employed in industry, particularly tem [17, 20, 21]. Exposure of BPA and BPS to zebrafish in polycarbonate plastics industrial processes and food- embryo causes 180 and 240% increase in hypothalamic stuff containers. The resiliency of BPA plastics has led to neurogenesis [21]. During early developmental stages, their use in medical equipment’s such as heart-lung ma- BPA exposure has been suspected to affect testicular de- chines, incubators, hemodialyzers, and dental sealants velopment and spermatogenesis [22, 23]. and fillers; also, their light weight and optical clarity have In mammals, ovaries are generally quiescent in devel- made them especially useful for eyeglasses. oping females, so reproductive tract and brain develop- help make plastic, like pacifiers, flexible. Due to his ment occurs in the absence of . Whereas in widespread applications, the use of BPA has gathered developing male, elevated estrogen levels locally synthe- cumulative consideration over the last decade, particu- sized by aromatization of (testicular) are larly in terms of human safety. It is believed that both present. The sexual differentiation of estrogen exposure BPA and phthalates can leach from the plastic into food, and hormonal synthesis results into distinct develop- liquid, and directly into the mouths of children while ment of neuroanatomical circuits, neuroendocrine func- sucking on pacifiers or teethers. It has been estimated tions, and reproductive behaviors in both male and that levels of conjugates of BPA in urine are above safety females [24]. The anteroventral periventricular nucleus thresholds in 90% of individuals tested in several popula- (AVPV) is a small cluster of neurons along the wall of tion studies [1]. Fetal exposure to high doses of dibutyl the third ventricle just caudal to the vascular organ of was shown to cause a testicular dysgenesis lamina terminalis (OVLT). AVPV receives sexually di- syndrome (TDS)-like phenotype in the rats [2]. TDS is a morphic innervation by dopaminergic afferents that male reproduction-related condition characterized by regulate gonatropin-releasing hormone and sexual the presence of symptoms and disorders such as hypo- reproduction [25, 26]. AVPV is three times more sexu- spadias, cryptorchidism, poor semen quality, and testicu- ally dimorphic in females, being larger in volume and lar cancer. TDS is a result of disruption of embryonal containing more cells in females than male. This sug- programming and gonadal development during fetal life gests that this brain region is very important in control- [2] Various reports established that BPA acts as an endo- ling the estrous cyclicity in females. In female AVPV, for crine disrupting chemical and its exposure can affect the dopamine synthesis, the rate limiting enzyme tyrosine reproductive system of a male by disturbing spermato- hydroxylase (TH) expressing neurons are more abun- genesis and fertility, furthermore, its exposure during de- dantly and topographically distributed and are distinct velopment causes organizational effects on brain [3–10]. from males [26, 27]. The perinatal administration of tes- BPA has increased excess to estrogen sensitive tissues in tosterone or estrogen can defeminize the neuron count brain [16]. At the hypothalamic or pituitary level, BPA and distribution in female AVPV [28]. One of the func- may inhibit the estrogen binding to its receptors. Thus, tion of TH cells of AVPV is to regulate the secretion of circulating estrogen reduces its negative feedback actions gonadotropin releasing hormone (GnRH) neurons in the on luteinizing hormone (LH) and follicle stimulating medial (POA) [29]. Total numbers of TH- hormone (FSH) release, resulting in high levels of circu- cells are more in female’s AVPV than males, hence sexu- lating LH and FSH. Thus, it is suggested that BPA’s ac- ally dimorphic GnRH secretions. Estradiol administra- tions are greater during development[13–15]. tion have no effect on TH cell numbers in males and it Bisphenol S (4, 4′–dihydroxydiphenyl sulphone) abbre- is suggested that BPA have action on this viated as BPS, is a man made, industrial chemical and an- neural cell population [24]. other member of bisphenol family. BPS has increased If it is speculated that BPA acts as estrogen or an anti- stability and resistance against sunlight and high tempera- estrogen in the AVPV region, then it can be hypothe- tures. Like BPA, BPS is an endocrine disrupting molecule sized that more or less sexually dimorphic areas could and its increasing use is alarming for human health [11]. be affected by exposure to BPA or BPS. Considering our In zebrafish, BPS exposure has shown to induce a reduc- chosen endocrine active compounds to be estrogenic, tion in gonadal weight, alteration in hormonal and disrup- masculinization of female AVPV should be evident via a tion in the normal process of reproduction (i.e. decreases reduction in TH-expressing neurons number. In con- egg production and hatchability, increases embryo malfor- trast, anti-estrogenic bisphenols through increment in mations, increase in time to hatch) [17]. BPS exposure TH-expressing neurons count should de-masculinized also decreases body length, increases male and female sex the male AVPV. ratio and causes reproductive disruption, disturbs the This study was designed to determine the low dose ef- balance of sex hormones in adult zebrafish [18]. fect of BPA and BPS on sexual differentiation of the Several studies show androgenic and anti-androgenic AVPV region of the male brain at the neonatal stage and activities of BPS [19]. Similar to BPA, BPS can induce to find out the possible role of endocrine disrupting John et al. Reproductive Biology and Endocrinology (2019) 17:53 Page 3 of 10

chemicals on the reproductive system of male rats at ini- Histological analysis tial stages of life. These finding will further provide the Histological analysis was done exactly by using a method evidence of negative effects of BPA and its analogue BPS given by Ullah et al. [34]. Firstly, testis was kept in sera on the reproductive system of male rats. (composed of ethyl alcohol, formaldehyde, and glacial acetic acid in a ratio of 6:3:1), and then placed in 10% formalin for 24 to 48 h. Following fixation, tissues were Methods dehydrated with ascending grades of ethanol, cleared Animals with xylene and embedded in paraffin wax. Microtomy At the start, five wooden breeding cages were separated was then carried out and seven μm thick sections of and five adult female Sprague Dawley rats were kept in testis were cut (Thermo, Shandon finesse 325, UK). each breeding cage with two adult male Sprague Dawley Testis sections were then fixed on albumenized glass rats. Ten days later, adult male rats were separated from slides, placed in an incubator overnight for completion females. Laboratory pelleted food and water was avail- of deparaffinization and on next day, were stained with able to animals ad libitum. Pregnant females were reared hematoxylin and eosin and then, observed under the singly till the birth of pups on day 22 (gestational day- light microscope (Nikon, 187,842, Japan). Leica LB GD-22). The day of birth of the pups was called as post- microscope (Germany) equipped with canon digital cam- natal day 1 (PND1. Total numbers of pups were counted era was used for microphotography. For histolomorpho- and separated from female pups by measuring the ano- metric studies, Image J software was used for the genital distance (AGD) under a stereomicroscope. For measurement of testis parameters (National Institutes of each experimental group, six male pups were separated Health, Bethesda, MD, USA). from each litter. Animals were handled and sacrificed according to the guidelines provided by the Ethical Brain tissue fixation and processing Committee of Animal Sciences department, Faculty of Theentirehypothalamicblocksofthebrainwere Biological Sciences, Quaid-i-Azam university, Islamabad. placed in 4% paraformaldehyde overnight. Next day, these samples were dehydrated and cleared through different grades of ethanol and xylene follow embed- Experimental plan ding in paraffin wax. The tissue was then deparaffi- Newborn pups (PND1) animals were distributed into six nized with xylene and rehydrated in graded ethanol groups (n = 6/group). The first group served as control before being washed with twice-distilled water. Later, and received subcutaneous injection of olive oil (50 μl). hypothalamic blocks were cut into consecutive sec- The second and third group rats were treated with BPA tions of 7–10 μm thickness on a cryostat (Bright (2 μg/kg and 200 μg/kg) respectively. Fourth and fifth OTF 5000, A-M Systems, Sequim, Washington, USA; group male pups were injected with BPS (2 μg/kg and temperature − 25 °C) and preserved in an antifreeze 200 μg/kg) respectively. The sixth group was given with cryoprotectant solution (1% polyvinylepyrrolidone, 50 μg/kg of estradiol dissolve in olive oil as a standard to 30% ethylene glycol and 30% sucrose in PBS) at − a find development of dopaminergic tyrosine hydroxy- 20 °C until used for immunocytochemistry. lase neurons in AVPV regions. All treatments were dissolved in olive oil and were given from PND 1 to Immunocytochemistry PND 27 with reference to prior studies [30–32]. Es- Standard double immunocytochemistry protocol was tradiol treated group was only used to serve as a followed for the processing of hypothalamic sections. standard to find a development of dopaminergic tyro- Total numbers of TH cell bodies were immunolocalized sine hydroxylase neurons in AVPV of rodents’ hypo- in the mediobasal hypothalamic region using a cocktail thalamus. All dosages were adjusted daily according of primary antibodies directed against TH. For TH ex- to body weight. We selected a higher dose of 200 μg/ pression monoclonal antibody TOH A1.1 raised in kg because it is higher suspected human exposure mouse against human TH (Catalogue no. ab-150,659; dose and a low dose of 2 μg/kg approaches to human Abcam Biotechnology, Inc., Cambridge, United King- exposure level [24, 33]. dom) was used and Alexa Flour 488 labeled goat anti- On PND 27, animals were weighed (using Sarotoreious mouse IgG (Catalogue no. ab150117; Abcam, Cam- Digital Balance) and sacrificed by decapitation. The bridge, UK) was used as a secondary antibody. From brain tissues were used only for immunohistochemistry. each animal, three slides of hypothalamus were obtained Testicular tissues were dissected out for tissue histology for TH labeled immunostaining. While, one section from and were washed in saline and weighed. Other tissues each group was used as primary antibody omitted con- (brain, kidney, liver, and intestine) were also dissected trol. Hypothalamic sections were washed with phosphate out and weighed also. buffered saline (PBS; PH7.3) (Omnipur PBS tablets, John et al. Reproductive Biology and Endocrinology (2019) 17:53 Page 4 of 10

Calbiochem, EDM chemicals Inc., Gibbstown, New Results Jersey, USA) for 8 × 15 minutes at room temperature Effect of subcutaneous exposure of bisphenol A (25 °C), to remove the cryoprotectant afterwards sections bisphenol S on body weight (g) in male rats, during the were incubated in incubation solution containing 10% neonatal period normal goat serum, 0.05% Triton-X 100 and 0.1% bovine Mean ± SEM body weight of all experimental groups in serum albumin (BSA) in PBS for two hours on shaker at male rats are shown in Table 1. As compared to control room temperature to block the non-specific binding of group, significant (p < 0.05) change was detected in body the antibodies. Sections were then washed with PBS for weight recorded on PND 8, among low and high con- 3 × 15 minutes. Then sections were incubated at 4 °C for centrations of BPA groups. On PND 16, very remarkable 48 h on a shaker in a cocktail of primary antibodies increment (p < 0.001) was noticed in body weight among (anti-TH antibody at 1:20) diluted at PBS containing BPA 2 μg/kg and BPS 2 μg/kg treated groups comparison 10% normal goat serum, 0.03% Triton X- 100 and 0.5 with control. Animals treated with BPS 200 μg/kg BSA. Control sections were incubated in PBS with 10% showed significance change (p < 0.05) than control normal goat serum in PBS containing 0.05% TritonX- group. BPA (2 μg/kg dose) induced significant (p < 0.01) 100 and 0.1% BSA. After incubation of 48 h, sections increase in body weight on PND 24 compared to the were washed in PBS for 3 × 15 min at room temperature. control group. After washing, the sections were incubated in the cocktail of secondary antibodies Alexa Flour 488 la- Effect of subcutaneous exposure of BPA and BPS on beled goat anti-mouse at 1:1000 diluted in PBS con- weight of testis (g), (g), seminal vesicle (g), taining 0.05% Triton X- 100 and 0.1 BSA 10% normal liver (g), heart (g) and kidney (g) goat serum for 2 h in dark, at room temperature on a Table 2 indicated the effect of various treatments of BPA shaker. Control sections were also incubated with sec- and BPS on organ weights of rats. ondary antibodies at this stage. After incubations, sec- Testicular weight seen to be increased (p < 0.05) in tions were again washed with PBS 3 × 15 min. Later, BPA 2 μg/kg, BPS 2 μg/kg and BPS 200 μg/kg treated all sections were mounted on super frosted glass groups than the control. No significant differences were slides (Micro slides, Santa Cruz Biotechnologies, Dal- noticed in prostrate, seminal vesicle, liver and kidney las, Texas USA) and left overnight for drying at 4 °C weights when all experimental groups were compared to in dark. Then coverslip was placed on slides using control. Heart weight of BPA and BPS 2 μg/kg and 2 μg/ laboratory-prepared gelvatol as a mounting medium. kg treated animals show significance change (p < 0.01) as Gelvatol was prepared by adding 10.5 g polyvinyl alco- compared to control. hol and few crystals of sodium azide in 12 ml gly- cerol. Then 21 ml distilled water and 53 ml Tris (Ph Tissues histology 8.5) were added. The mixture was stirred on low heat The histological studies of the testis showed closely ar- for six hours until reagents were properly dissolved. ranged seminiferous tubules and normal spermatogen- The mixture was placed overnight in a refrigerator esis in the control group. A photomicrograph of a and centrifuged at 5000 g for 15 minutes. Slides were section of the testis of 27 days old albino rat of the BPA stored at 4 °C after drying until further analysis. Slides 2 μg/kg group showing a prominent increase in the were viewed using fluorescent microscope (Bx51, interstitial spaces between seminiferous tubules ap- Olympus, Tokyo, Japan) to localize, Tyrosine hydroxy- peared irregular and smaller in size as compared to con- lase immuno-reactive neurons in the AVPV region of trol group. A decrease was observed in the epithelial the brain. Sections were examined at 10X, 20X and diameter so it is evident that there will be a reduction in 40X magnifications. the number of spermatogonia and primary spermato- cytes near the lumen of the tubule. Some germ cells ap- peared shattered and separated with the appearance of Statistical analysis empty spaces; some appeared desquamated toward the For histological data analysis of testes and the number lumen with the disappearance of the early spermatids. of single TH-immunoreactivity cells, GraphPad prism 5 Lumen diameter in BPA 2 μg/kg treated group was in- software (GraphPad Software, Inc., San Diego, CA, creased as compared to the control group. BPA 200 μg/ USA) was used. One way analysis of variance (ANOVA) kg treated rat testis showing a normal testis structure was used for statistical analysis of studied parameters. with a slight decrease in tubular diameter as compared Later, Dunnet’s multiple comparison tests were prac- to BPA 2 μg/kg. There was an increase in the interstitial ticed to relate the controls results with treated ones. All spaces between the tubules with a slight appearance of thedataisshownasmean±SEM.Significancevalue destructed Leydig cells. BPA 200 μg/kg treated rats was set at p <0.05. showed less loss of stratification and disorganization of John et al. Reproductive Biology and Endocrinology (2019) 17:53 Page 5 of 10

Table 1 Comparative effects of different concentrations (2 μg/kg, 200 μg/kg) of Bisphenol A and Bisphenol S on body weight (g) recorded on 8th, 16th, 24th and 28th day of development Groups Day 8 Day 16 Day 24 Day 28 Control 9.71 ± 0.52 12.4 ± 0.87 20.5 ± 0.92 43.7 ± 6.54 BPA (2 μg/kg) 13.6 ± 0.65 21.9 ± 0.32 a** 35.3 ± 0.60 a** 40.1 ± 1.66 BPA (200 μg/kg) 13.0 ± 0.30 12.8 ± 0.87 19.4 ± 1.88 37.6 ± 3.38 BPS (2 μg/kg) 11.5 ± 0.28 19.9 ± 1.50 a**c*** 28.9 ± 2.79 40.4 ± 0.32 BPS (200 μg/kg) 13.9 ± 0.38 17.2 ± 0.41 a* 31.6 ± 2.12 a*c*** 38.5 ± 2.05 Values are expressed as mean SEM *p < 0.05, **p < 0.01, p*** < 0.001 a = Values vs control, b = Values vs Bisphenol A 2 μg/kg, c = Values vs Bisphenol A 200 μg/kg, d = Values vs Bisphenol S 2 μg/kg the lining epithelium of the seminiferous tubules show Epithelial height more closely resembled to control group. Epithelial layer Mean epithelial thickness was decreased significantly contained spermatogonia, primary spermatocytes, and (p < 0.001) in BPA 2 μg/kg treated group as compare to early spermatids. Some interfollicular spaces of the tu- control group. Highly significant (p < 0.001) reduction bules showed destructed interstitial tissue and most sec- was seen in BPA 200 μg/kg treated group as compare to tions of testis treated with BPA 200 μg/kg shows a control group. There was highly significant (p < 0.001) reduction in lumen diameter show slight variation as decrease in mean was observed between BPS 200 μg/kg compare to control group. Histomorphology of the treated group and BPS 2 μg/kg treated group as compare testicular section from BPS 2 μg/kg showed a major dif- to control group (Table 3, Fig. 1). ference in the appearance of seminiferous tubules com- pared to control group. Lumen diameter is somehow Tunica Albugenia height appeared similar to the control group. There was no significant change was noticed in Tunica Albuginea height among all treated groups except BPA Seminiferous tubule diameter 2 μg/kg showed significant decrease (p < 0.005) with Significantly increased (p < 0.001) diameter was observed their comparison to that of the control group (Table 3, when BPA 2 μg/kg and BPS 200 μg/kg treated groups Fig. 1). compare with a control group. No significant difference was found when compared BPS 2 μg/kg treated group TH immunoreactive cells with BPA 200 μg/kg treated groups as compared to con- TH expression is sexually dimorphic in the AVPV region trol. (Table 3, Fig. 1). of brain. No significant alterations were observed in TH- ir cell number between control, estradiol and a low dose Tubular lumen diameter of BPA 2 μg/kg treated groups. Extremely prominent Mean tubular lumen diameter in BPA treated animals (p < 0.001) rise was seen in the TH-ir cell bodies between was decreased significantly (p < 0.001) as compare to BPA 200 μg/kg treated group and control group. Treat- control animals. Statistical difference in the mean of the ment with a low dose of BPS 200 μg/kg and a high dose lumen diameter was decreased significantly (p < 0.01) of BPS 2 μg/kg were significantly (p < 0.001) different when compare BPA 200 μg/kg treated group with a con- from control group (Table 4, Fig. 2). trol group. A highly significant decrease (p < 0.001) in tubular lumen diameter was observed in BPS 2 μg/kg Discussion and BPS 200 μg/kg treated groups respectively when Over the last decade, it has become well known that compared to control group (Table 3, Fig. 1). BPA, a ubiquitous environmental endocrine disruptor

Table 2 Comparative effects of different concentrations (2 μg/kg,200 μg/kg) of Bisphenol A and Bisphenol S on body organs weight Groups Testes paired weight(g) Prostate (g) Seminal Vesicle (g) Liver (g) Heart (g) Kidney (g) Control 0.74 ± 0.19 0.04 ± 0.01 0.34 ± 0.19 1.90 ± 0.20 0.58 ± 0.22 0.59 ± 0.21 BPA 2 μg/kg 0.24 ± 0.01a* 0.06 ± 0.01 0.03 ± 0.01 1.90 ± 0.17 0.20 ± 0.01** 0.45 ± 0.01 BPA 200 μg/kg 0.39 ± 0.17 0.05 ± 0.01 0.01 ± 0.0 1.92 ± 0.08 0.23 ± 0.02** 0.49 ± 0.02 BPS 2 μg/kg 0.25 ± 0.01 0.02 ± 0.01b** 0.09 ± 0.07 1.63 ± 0.13 0.23 ± 0.01** 0.55 ± 0.00 BPS 200 μg/kg 0.24 ± 0.03 0.02 ± 0.01b** 0.02 ± 0.01 1.43 ± 0.15 0.23 ± 0.02** 0.47 ± 0.02 Values are expressed as mean SEM *p < 0.05, **p < 0.01, p*** < 0.001 a = Values vs control, b = Values vs Bisphenol A 2 μg/kg, c = Values vs Bisphenol A 200 μg/kg, d = Values vs Bisphenol S 2 μg/kg John et al. Reproductive Biology and Endocrinology (2019) 17:53 Page 6 of 10

Table 3 Mean ± SEM of seminiferous tubule diameter, tubular lumen diameter, seminiferous tubule epithelial height, tunica albuginea height of testis in control and treated groups after 28 days of treatment Groups Seminiferous Lumen Epithelial Height Tunica Albuginea Tubule Diameter Diameter Height Control 106.46 ± 2.94 84.19 ± 3.46 35.59 ± 0.63 19.79 ± 1.14 BPA 2 μg/kg 126.03 ± 4.19a*** 61.72 ± 3.09 a*** 23.00 ± 0.48 a*** 16.34 ± 0.54 BPA 200 μg/kg 133.98 ± 3.23a*** 71.22 ± 2.60 a** 25.16 ± 0.48ab*** 17.66 ± 0.90 BPS 2 μg/kg 106.60 ± 2.46bc*** 62.90 ± 1.77 a*** 32.39 ± 0.54abc*** 18.61 ± 0.63 BPS 200 μg/kg 114.29 ± 2.45c*** 64.86 ± 2.20 a*** 26.55 ± 0.57abd*** 18.19 ± 0.67 Values are expressed as mean SEM *p < 0.05, **p < 0.01, p*** < 0.001 a = Values vs control, b = Values vs Bisphenol A 2 μg/kg, c = Values vs Bisphenol A 200 μg/kg, d = Values vs Bisphenol S 2 μg/kg administration causes reproductive toxicity and interstitial spaces and tubular lumen diameter with expos- gonadal damage [35–39]. Similarly, BPS is another ure to bisphenol analogues. Both BPA low and high dose strong estrogenic and anti-androgenic compound, exposure led to reduce seminiferous tubule diameter, epi- which has been banned now in different countries due thelial and tunica albugenia height and germ cells. In our to its toxic effects on the physiology of the reproduct- former study by Jahan et al., 2016, it has been reported ive system [19, 40, 41]. that administration of BPA in adult rats induces similar The present study shows irreversible organizational ef- changes with low efficiency of spermatogenesis. Studies fects in testis of prepubertal male rats exposed to BPA have shown that not only tissue morphology is affected by and BPS different concentrations (2 μg/kg and 200 μg/ BPA but also the number of mature spermatids becomes kg) during the neonatal stage [42]. We observe no sig- limited [34]. Similarly, low dose exposed BPS groups nificant change in final body weight in all treatment underwent a minimal level of damage to epithelial and re- groups. These results are similar to a study where ani- duction in lumen diameter, however, seminiferous tubule mals were treated with 50 μl and 5 μg/kg BPA presented almost same pattern as control and as such no (Gen/BPA 0.005) and no significant change in body evident damage in high dose BPS exposed group. weight were observed [43]. Alike results were experi- In vertebrates and mammals, reproductive system and enced in postnatal male mice as compared to control brain are physiologically and anatomically differentiated mice when administered with BPA at early embryonic in both males and females, as a result of postnatal sex stage [36]. Ullah et al. (2016) also noticed no alteration during early development in the hypothalamic in body weight of adult male rats after sub-chronic oral sexually differentiated anteroventral periventricular administration of BPS [34]. However, in the current nucleus (AVPV). AVPV region of the brain has multiple study significant increase in body weights was recorded cell types that are sexually dimorphic and tyrosine at PND 16 and PND 24 in BPA (2 μg/kg) and BPS (2 μg/ hydroxylase positive cells are one of them [47]. kg and 200 μg/kg) treated groups. Our results are in ac- Aromatization of estrogen in the medial region of male cordance with the former study by Rubin et al. (2001) brain results in expression of few Tyrosine hydroxylase where female rat dams, when exposed to BPA from day cell. It is well documented that in rats and mice, neurons 6 of pregnancy until lactation, resulted in increased body of TH + ve cells in the AVPV of brain act as a potent weight relative to control group in offspring [45]. Previ- anatomical marker of brain sexual differentiation [48, ously, Sakaue et al. reported that exposure of BPA (2 μg/ 49]. TH neurons number in the AVPV region of brain is kg to 200 μg/kg) to adult male rats resulted in decreased effectively reduced by administrating testosterone or es- testicular weight and impaired spermatogenesis [44]. tradiol at the perinatal and/or postnatal stage. No sex It is known that administration of exogenous estrogen difference in TH number was seen in ERKO (lack ER) results in reduced adipocyte number and it might con- mice, however, mice lacking TH receptor for androgen tribute to reduction of body weight [46–50]. No signifi- maintain sexual dimorphism in TH number [49]. So cant change was seen for liver, kidney and heart weights through these studies, it might be proposed that ER is when compared in experimental groups (Table 2). Some important for estrogen action in reducing TH neuron studies showed no significant change in organ weights number in males as compared to females. by BPA administration [39]. The postnatal exposure of bisphenol analogues has In the present study, pubertal spermatogenesis for histo- displayed no significant effect at low dose 2 μg/kg of morphometric analysis was characterized by seminiferous BPA and BPS on the total amount of TH-ir neurons in tubule diameter, lumen diameter, epithelial and tunica males. Our present results are in line with findings of albugenia height. A significant increase was witnessed in Patisaul et al. (2006) [24]. But the significant increase John et al. Reproductive Biology and Endocrinology (2019) 17:53 Page 7 of 10

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E Fig. 1 Photomicroghaph of 27 days old neonatal male rat seminiferous tubules(H&E, 40X) from: (a) Control; showing normal morphology of closely packed tubules with basal lamina, stratification and increased spermatogenic epithelium Spermatogonia, primary spermatocytes and early spermatids well developed lumen, (b) BPA group treated with 2 μg/kg of dose; showing noticeable increase in interstitial spaces between destructed tubules and basal lamina, decrease in epithelial height, only spermatogonia and large lumen, (c) BPA group treated with 200 μg/kg; showing decreased tubular diameter damaged interstitial tissues and increased interstitial spaces, narrow lumen, (d) BPS 2 μg/kg treated group showing semineferious tubules without interstitial space, minimal damage to epithelial, decrease in lumen diameter, (e) BPS 200 μg/kg; showing no interstitial space, normal spermatogenesis and basal lamina around tubules and very short lumen with early spermatids. Interstitial space (IS), Spermatogonia(S), Primary spermatocytes (PS), Early spermatids (ES), Epithelial Height (EH), Interstitial tissues (IT), Basal lamina (BL), Lumen (L) was observed in the number of TH-ir cell bodies with expression of TH in AVPV region suggesting that high doses of 200 μg/kg of BPA and BPS. These re- BPA, as well as BPS, play an anti-estrogenic role in sults indicate that BPA interferes with estrogen action the developing brain of male rats. Various scientists at low dose [28] and acts as antiestrogenic at high also support this idea [24]. doses resulting in demasculinization of positive cells The underlying mechanisms behind estrogen actions that can lead to reproductive damage in adulthood. on TH expression patterns in AVPV in the neonatal The data point that just like BPA, BPS effectively in- males are not well understood. But it appears to be act- terferes with endogenous estrogen and demasculinizes ing through signaling. According to John et al. Reproductive Biology and Endocrinology (2019) 17:53 Page 8 of 10

Table 4 Comparison of Mean ± SEM TH-ir neuronal cells of control, estradiol, BPA 2 μg/kg, BPA 200 μg/kg, BPS 2 μg/kg, BPS 200 μg/ kg treated groups, in 20 μm thick hypothalamic sections of 28 days old male rats Animals Control Estradiol 50 μg/kg BPA 2 μg/kg BPA 200 μg/kg BPS 2 μg/kg BPS 200 μg/kg 1 74.5 60.75 75 163.75 183 148 2 46.25 56.25 101 178.25 100.75 119.5 3 65.25 56.5 94.75 56.5 64.5 87 TH-ir Cell Bodies 62 ± 4.47 57.83 ± 3.68 90.25 ± 7.41b* 175 ± 8.2abc*** 80.08 ± 10.03d*** 118.16 ± 10.23abd***e* Values are expressed as mean SEM *p < 0.05, **p < 0.01, p*** < 0.001 a = Values vs control, b = Values vs Bisphenol A 2 μg/kg, c = Values vs Bisphenol A 200 μg/kg, d = Values vs Bisphenol S 2 μg/kg

Rubin et al. (2011), a study conducted on the pregnant suggesting that ERa is important for the normal mice delivering low levels of BPA to their offspring by masculinization of the male brain by estrogen [44–49]. subcutaneous implanted Alzet pumps resulted into de- It is also demonstrated that developing AVPV TH-ir creased TH cell number in AVPV of females as com- cells are sensitive to EACs disruption, reliability of dopa- pared to control male [12]. Simerly et al., (1997) minergic TH expression patterns make it more vulner- reported that male mice in which TH-ir neurons lack able to examine the sensitivity of EACs in the neonatal ERa, are phenotypically similar to wild type female mice, brain [50]. Previous epidemiological studies for early life

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Fig. 2 Photomicrograph (10X) of TH-immunoreactive cells in AVPV of representative Sprague Dawley male rat pups that were neonatal treated with BPA 2 μg/kg (a), BPA 200 μg/kg (b), BPS 2 μg/kg (c), BPS 200 μg/kg (d), Control (e), Estradiol (f). Omitted controls g and h show no significance results John et al. Reproductive Biology and Endocrinology (2019) 17:53 Page 9 of 10

exposure of BPA in young girls in humans associated Ethics approval and consent to participate with a high level of anxiety and hyperactivity suggest This study makes use of rats, and the experimental protocol for the use of animal was approved (BAS#0256) by the ethical board of Quaid-i-Azam Uni- that early life BPA exposure has injurious, sex specific versity, Islamabad Pakistan. and neural effects in humans [51, 52, 56]. According to observations of the USA population from Consent for publication 2010 to 2014, a trend of using BPS as BPA substitute Not applicable. has led to increased use of this chemical in industrial products [53]. Sexual dimorphism in AVPV volume and Competing interests TH neurons are appeared to be influenced by BPA ex- The authors declare that they have no competing interests. posure and there are different mechanisms that appear Received: 14 March 2019 Accepted: 10 June 2019 to be responsible to influence both, including cell surviv- ing gene Bcl-2 and BAX gene deletion and overexpres- sion, they are responsible for cell death reduction in References 1. Anway MD, Skinner MK. Epigenetic transgenerational actions of endocrine AVPV and eliminated sex differences in AVPV volume disruptors. Endocrinology. 2006;147:s43–9. in mice [54–57]. However, all these manipulations were 2. Arnich N, Canivenc-Lavier M-C, Kolf-Clauw M, Coffigny H, Cravedi J-P, Grob failed to alter the TH neuron number in this nucleus. K, Macherey A-C, Masset D, Maximilien R, Narbonne J-F. Conclusions of the French food safety agency on the toxicity of bisphenol A. 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